Modified polypeptides with altered biological activity

Abstract

The invention relates to novel modified polypeptides, with or without variations in noncoding regions, with altered biological activity. The invention discloses methods of preparing the modified polypeptides and methods of use.

What is claimed is: 
     
       1. A biologically active polypeptide homodimer wherein each polypeptide of the homodimer comprises a four alpha helical bundle polypeptide and the plasma half-life of the homodimer increased in comparison to wildtype monomer, comprising two polypeptides covalently linked by a least one thioether bond, wherein: a) the first polypeptide comprises a polypeptide with a heterobifunctional cross-linking reagent containing a free sulfhydryl group attached to the polypeptide; and   b) the second polypeptide comprises a polypeptide with a heterobifunctional cross-linking reagent containing a maleimido group attached to the polypeptide and at least one thioether bond forms between the free sulfhydryl group of the first polypeptide and the maleimido group of the second polypeptide.   
     
     
       2. The polypeptide homodimer of claim 1 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of: N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproic acid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester, and β-maleimidoproprionic acid N-hydroxysuccinimide ester. 
     
     
       3. A composition comprising a polypeptide homodimer according to claim 1 and a pharmaceutically acceptable carrier. 
     
     
       4. A biologically active polypeptide homotrimer wherein each polypeptide of the homotrimer comprises a four alpha helical bundle polypeptide and the plasma half-life of the homotrimer increased in comparison to wildtype monomer, comprising three polypeptides covalently linked by thioether bonds, wherein: a) the first and second polypeptides comprise polypeptides with a heterobifunctional cross-linking reagent containing a free sulfhydryl group attached to each polypeptide; and   b) the third polypeptide comprises a polypeptide with a heterobifunctional cross-linking reagent containing two, or more, maleimido groups attached to the polypeptide and the thioether bonds form between the free sulfhydryl group of the first and second polypeptides and the maleimido groups of the third polypeptide.   
     
     
       5. The polypeptide homotrimer of claim 4 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of: N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproic acid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester, and β-maleimidoproprionic acid N-hydroxysuccinimide ester. 
     
     
       6. A composition comprising a polypeptide homodimer according to claim 4 and a pharmaceutically acceptable carrier. 
     
     
       7. A biologically active polypeptide homotrimer wherein each polypeptide of the homotrimer comprises a four alpha helical bundle polypeptide and the plasma half-life of the homotrimer increased in comparison to wildtype monomer, comprising three polypeptides covalently linked by thioether bonds, wherein: a) the first polypeptide comprises a polypeptide with a heterobifunctional cross-linking reagent containing two, or more, free sulfhydryl groups attached to the polypeptide; and   b) the second and third polypeptides comprise polypeptides with a heterobifunctional cross-linking reagent containing a maleimido group attached to each polypeptide and the thioether bonds form between the free sulfhydryl groups of the first polypeptide and the maleimido group of the second and third polypeptides.   
     
     
       8. The polypeptide homotrimer of claim 7 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of: N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproic acid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester, and β-maleimidoproprionic acid N-hydroxysuccinimide ester. 
     
     
       9. A composition comprising a polypeptide homodimer according to claim 7 and a pharmaceutically acceptable carrier. 
     
     
       10. The polypeptide homodimer of claim 1 wherein the heterobifunctional cross-linking reagents of a) and b) are attached to one, or more, primary amine or amines in the polypeptides. 
     
     
       11. The polypeptide homodimer of claim 1 wherein the polypeptide is glycosylated and the heterobifunctional cross-linking reagents are attached to one, or more, carbohydrate moiety or moieties in the glycosylated polypeptides. 
     
     
       12. A method of preparing a biologically active polypeptide homodimer wherein each polypeptide of the homodimer comprises a four alpha helical bundle polypeptide and the plasma half-life of the homodimer increased in comparison to wildtype monomer, comprising the steps consisting of: a) preparing a first polypeptide derivative by reacting polypeptide with a heterobifunctional cross-linking reagent containing a cleavable disulfide bond group, under conditions sufficient to introduce the cleavable disulfide bond group into the polypeptide, thereby producing a first polypeptide derivative containing a cleavable disulfide bond;   b) cleaving the disulfide bond group contained in the first polypeptide derivative, thereby producing a first polypeptide derivative containing a free sulfhydryl, group;   c) preparing a second polypeptide derivative by reacting polypeptide with a heterobifunctional cross-linking reagent containing a maleimido group, under conditions sufficient to introduce the maleimido group into the polypeptide, thereby producing a second polypeptide derivative containing a maleimido group; and   d) reacting the first polypeptide derivative containing a free sulfhydryl group with the second polypeptide derivative containing a maleimide group, under conditions sufficient to form a thioether bond between the free sulfhydryl group and the maleimido group resulting in the cross-linking of the first and second polypeptide derivatives, thereby producing a modified polypeptide comprising a first and second polypeptide derivative.   
     
     
       13. The polypeptide homodimer of claim 12 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of: N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain" N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated "long chain" succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproic acid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester, and β-maleimidoproprionic acid N-hydroxysuccinimide ester. 
     
     
       14. The method of claim 12 wherein the heterobifunctional cross-linking reagents of step a) and step c) react with one, or more, primary amine or amines in the polypeptide. 
     
     
       15. The method of claim 12 wherein the polypeptide is a glycosylated polypeptide and the heterobifunctional cross-linking reagents of step a) and step c) react with one, or more, carbohydrate moiety or moieties in the glycosylated polypeptide. 
     
     
       16. A composition comprising a polypeptide homodimer according to claim 12 and a pharmaceutically acceptable carrier.